Co-doped carbon dot nano-enzyme particle, preparation method, detection test paper and application

Abstract

The level of alkaline phosphatase (ALP) in a whole blood sample is monitored by using cobalt-doped carbon dot (Co-CDs) nano-enzyme with excellent oxidase simulation catalytic activity and red fluorescence emission and combining with A-3D [mu] PADs; co-CDs catalyzes the oxidation of non-fluorescent OPD into oxOPD with yellow fluorescence emission, and a large amount of O2 <.-> substances are generated; after ALP is added, AAP is hydrolyzed into AA by ALP, the AA is oxidized by Co-CDs and then reacts with OPD to generate DFQ emitting blue fluorescence; both the DFQ and the oxOPD can quench the fluorescence intensity of the Co-CDs through an inner filtering effect; a portable micro device and a smart phone are integrated, and the ALP detection device is used for detecting ALP simply, conveniently and visually. The detection result of the method on a clinical sample is consistent with the detection value of a biochemical analyzer. The A-3D [mu] PAD can be used for measuring various biomarkers due to high efficiency, disposable use, practicability and low cost, and a technical support is provided for the application of the nano-enzyme in a POCT (Point of Care Testing) environment.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials and medical chemical analysis, and in particular to Co-doped carbon dot nanoparticles with oxidase-like activity, and a method for real-time detection of alkaline phosphatase activity thereof. Background Art

[0002] Alkaline phosphatase (ALP) is a key hydrolase that catalyzes the dephosphorylation of biological molecules such as nucleic acids and proteins. Elevated ALP levels are closely related to liver diseases, bone diseases, diabetes, cancer, etc., while decreased ALP levels are related to leukemia, thyroid diseases, etc.

[0003] A variety of methods for monitoring ALP activity have been developed, including electrochemistry, colorimetry, fluorescence, chemiluminescence, and surface-enhanced Raman scattering-based techniques. Among them, colorimetry and fluorescence are widely used due to their high sensitivity and ease of operation. However, the results of colorimetry are easily affected by the color of other substances in biological fluids, such as red blood cells. Therefore, fluorescence methods have attracted much attention due to their simplicity, sensitivity, and high throughput. At the same time, the above methods also face huge challenges, such as complex sample preparation, sophisticated instruments, and well-trained operators.

[0004] Compared with laboratory-based tests, POCT (point-of-care testing) has attracted much attention due to its advantages of intelligence, simple operation and digital readout. Paper-based microfluidic chips (microfluidic paper-based analytical device, μPAD) are potential candidates for POCT due to their advantages such as easy availability of paper, easy manufacturing, strong capillary driving force and storage capacity of reagents. Among them, 2D μPADs are composed of hydrophilic channels (such as chromatography paper, filter paper, etc.) and hydrophobic barriers (such as wax / laser / thermal printing, drawing, etc.), and 3D μPADs are prepared by folding, layering and stacking of 2D μPADs. The sample extraction and bioanalysis properties of 2D and 3D μPADs provide a powerful platform for clinical and biomedical applications. However, μPADs generally have the problem of low detection sensitivity, and researchers are committed to amplifying visual detection signals to improve sensitivity. The introduction of new materials (e.g., gold nanoparticles, nanocontainers, etc.), sample pre-enrichment techniques, multiple cascade reactions, and more sensitive sensing strategies (e.g., fluorescence, chemiluminescence, etc.) have been shown to effectively improve the detection sensitivity of μPADs. Changing the structure of 2D μPADs to 3D μPADs with multiple analysis capabilities can also improve sensitivity. In our previous study, folded and syringed 3D μPADs have been successfully used in the detection of ALP. Therefore, μPADs provide a sensitive, accurate, and easy-to-operate POCT platform for ALP detection, especially for communities with limited resources, promoting the development of precision medicine and personalized medicine.

[0005] Compared with natural enzymes, nanozymes with enzyme-mimicking properties have the advantages of easy preparation, high catalytic efficiency, high stability, and adjustable activity. Many nanozymes have been reported to have intrinsic oxidase, peroxidase, and catalase mimetic activities and have been applied in biosensing, biomedicine, food safety, and environmental protection. Materials with oxidase-like activities such as single-atom catalysts, metal-organic frameworks, carbon-based nanomaterials, and composite nanomaterials have been widely explored for the development of various biosensors. Carbon dots (CDs) are a typical carbon-based nanomaterial with excellent photoluminescence properties, low cytotoxicity, and good biocompatibility, and have been widely used in the field of sensing. Recently, it has been reported that CDs have oxidase-like activities and can be used to construct sensitive and accurate sensing platforms for the detection of nitrite, pesticides, and antibiotics. Metal doping (eg, Mn, Co, Pt doping) and surface modification (such as physical adsorption, covalent coating, etc.) have been shown to provide more binding sites, thereby significantly improving the catalytic activity of CDs. Through rational design, the oxidase-like activity of CDs avoids the introduction of external natural oxidases, and combined with the fluorescence properties, a light-responsive nanozyme sensing platform is realized.

[0006] Therefore, the development of a reliable, low-cost, large-scale equipment-free, and easy-to-operate POCT platform for the immediate detection of ALP activity is of great significance for promoting disease diagnosis, especially in home care and remote areas. Summary of the invention

[0007] The main purpose of the present invention is to provide Co-doped carbon dot nanoparticles with oxidase-like activity, and a method and application thereof for real-time detection of alkaline phosphatase activity, an alkaline phosphatase detection test strip, and a system for real-time detection of alkaline phosphatase, so as to realize rapid detection of alkaline phosphatase activity.

[0008] To achieve the above object, the present invention provides a method for preparing Co-doped carbon dot nanoparticles, characterized in that it comprises the steps of:

[0009] S1, dissolving neutral red, urea and cobalt chloride in pure water at a weight ratio of 0.8-1.2:0.8-1.2:1.8-2.2 and a mass-liquid ratio of 1.8-2.2:1;

[0010] S2, reacting the uniformly dispersed mixed solution in step S1 in a polytetrafluoroethylene-lined autoclave at 110° C. to 130° C. for 1.5 to 2.5 hours;

[0011] S3, dialyzing the reaction solution in step S2 for 36-60 hours;

[0012] S4, freeze-drying the solution obtained in step S3 to obtain Co-doped carbon dot nanoparticles.

[0013] Furthermore, the step S4 comprises filtering the solution obtained in step S3 through a filter membrane, and freeze-drying the filtered solution to obtain Co-doped carbon dot nanoparticles.

[0014] Furthermore, the pore size of the filter membrane is 0.2-0.24 μm.

[0015] Furthermore, the mass ratio of neutral red, chloroplatinic acid and ferric chloride in step S1 is 1:1:2.

[0016] Furthermore, the reaction temperature in step S2 is 115° C. to 125° C., and the reaction time is 1.8-2.2 h; and the dialysis time in step S3 is 45-55 h.

[0017] The present invention also provides Co-doped carbon dot nanoparticles, which are prepared by any of the preparation methods described above.

[0018] Furthermore, a use of the Co-doped carbon dot nanoparticles as described above for oxidizing ascorbic acid to dehydroascorbic acid in instant alkaline phosphatase activity.

[0019] The present invention also provides an alkaline phosphatase activity detection test strip, comprising a plasma separation membrane located at the top, a color developer layer located in the middle layer, and a color catalytic layer located at the bottom; the color developer layer comprises a first hydrophilic paper base region having a hydrophobic boundary and loaded with a color developer, and the first hydrophilic paper base region is located below the plasma separation membrane; the color catalytic layer comprises a second hydrophilic paper base region having a hydrophobic boundary and loaded with Co-doped carbon dot nanoparticles and L-ascorbic acid-2-phosphate prepared by any of the preparation methods described above; the first hydrophilic paper base region and the second hydrophilic paper base region are arranged to be connected.

[0020] Furthermore, the developer is non-fluorescent o-phenylenediamine.

[0021] The present invention also provides a system for real-time detection of alkaline phosphatase activity, comprising an alkaline phosphatase test strip as described in any of the above items, a dark box and an electronic device, wherein the dark box is provided with an ultraviolet lamp, a placement area for placing the alkaline phosphatase test strip and a shooting hole arranged corresponding to the placement area, the electronic device is used to take a photo of the alkaline phosphatase test strip after color development through the shooting hole, and the electronic device is used to analyze and compare the photo of the alkaline phosphatase test strip after color development through software to obtain a detection result of whether alkaline phosphatase is detected.

[0022] The present invention uses the cobalt-doped carbon dots (Co-CDs) / o-phenylenediamine (OPD) system and combines it with A-3DμPAD to develop a multi-emission fluorescence platform for visual detection of ALP in human whole blood. Neutral red (NR), urea, and CoCl2 are used as precursors, and red-light-emitting Co-CDs are prepared by a one-step hydrothermal method. Cobalt-doped carbon dots (Co-CDs) nanozymes have excellent oxidase-mimicking catalytic activity and red fluorescence emission, and are combined with a three-dimensional microfluidic paper chip (Adhered-3DμPADs, A-3DμPADs) to monitor the level of alkaline phosphatase (ALP) in whole blood samples. The Co(Ⅱ) active sites in Co-CDs provide excellent oxidase mimetic activity, catalyzing the non-fluorescent OPD to generate 2,3-diaminophenazine (oxOPD) with yellow fluorescence emission; in the presence of ALP, AAP is hydrolyzed to ascorbic acid (AA), AA is oxidized by Co-CDs to dehydroascorbic acid (DHAA), and DHAA reacts with OPD to generate 3-(dihydroxyethyl)furan-[3,4-b]-quinoxaline-1-one (DFQ) with blue fluorescence emission; both DFQ and oxOPD can quench the red fluorescence of Co-CDs through the inner filter effect (IFE). Based on the above sensing principle, an ALP monitor was constructed based on the Co-CDs / OPD system, and then these reactions were integrated into A-3DμPAD for real-time detection of ALP activity. As the ALP activity increases, the detection area of ​​the A-3DμPAD paper chip shows a clear red-yellow-blue fluorescence signal output. A-3DμPADs can realize sample pretreatment, multi-step cascade reaction and signal output on paper, collect corresponding images through smartphones, and convert them into red (R), green (G), and blue (B) values ​​through color recognition applications to obtain ALP activity. Thus, a feasible, sensitive, and low-cost on-site analysis platform for ALP activity was constructed by utilizing the superior oxidase-like activity of Co-CDs nanozymes and A-3DμPAD paper chips. The linear range and detection limit of ALP detection were 0.5-150U L -1 and 0.1UL -1 , the color of the paper base changed from red to yellow and blue. The test results of this method for clinical samples were consistent with the test values ​​of the biochemical analyzer. The high efficiency, disposable use, practicality and low cost of A-3DμPAD can be used to measure a variety of biomarkers, providing technical support for the application of nanozymes in POCT environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0024] Figure 1 A is a TEM image of Co-CDs prepared in Example 1 (inset: particle size distribution of Co-CDs); Figure 1 B is the XRD spectrum of Co-CDs prepared in Example 1; Figure 1 C is the UV-visible absorption spectrum and fluorescence excitation and emission spectrum of Co-CDs prepared in Example 1 (Inset: images of Co-CDs under sunlight and 365 nm UV light); Figure 1 D is the fluorescence spectrum of Co-CDs prepared in Example 1 at different excitation wavelengths (340-385 nm).

[0025] Figure 2 A is the FT-IR spectra of Co-CDs and CDs, Figure 2 B is the XPS measurement spectrum of Co-CDs and CDs, Figure 2 C is the high-resolution XPS spectrum of Co 2p of Co-CDs. Figure 2 D is the high-resolution XPS spectrum of O1s of Co-CDs. Figure 2 E is the high-resolution XPS spectrum of N1s of Co-CDs. Figure 2 F is the high-resolution XPS spectrum of C1s of Co-CDs.

[0026] Figure 3 A is the UV-vis spectra of CDs, CDs+OPD, Co-CDs, Co-CDs+OPD and OPD; Figure 3 B is the fluorescence spectra of CDs, CDs+OPD, Co-CDs, Co-CDs+OPD and OPD; Figure 3 C is the relative activity of Co-CDs catalyzing the OPD color development reaction at different pH; Figure 3 D is the UV-vis spectra of Co-CDs+TMB, Co-CDs+ABTS, Co-CDs+OPD, and Co-CDs; Figure 3 E is the picture of TMB, ABTS, and OPD solution before and after adding Co-CDs; Figure 3 F is the ESR spectrum of Co-CDs / DMPO system.

[0027] Figure 4 Schematic diagram of the structure of A-3DμPAD used to detect ALP activity.

[0028] Figure 5 A is a schematic diagram of the ALP sensing mechanism; Figure 5 B is the UV-vis spectrum of Co-CDs(1),ALP+AAP+Co-CDs(2),ALP+AAP+Co-CDs+OPD(3)AAP+Co-CDs+OPD(4); Figure 5 C is the fluorescence spectra of Co-CDs (1), ALP+AAP+Co-CDs (2), ALP+AAP+Co-CDs+OPD (3) and AAP+Co-CDs+OPD (4).

[0029] Figure 6 A is the fluorescence excitation and emission spectra of Co-CDs, and the UV-vis spectra of DFQ and ox OPD; Figure 6 B is the fluorescence lifetime of Co-CDs, Co-CDs+DFQ and Co-CDs+oxOPD; Figure 6 C is the Zeta potential test results of Co-CDs, DFQ and oxOPD.

[0030] Figure 7 The distribution of different volumes (10-20 μL) of Rhodamine B solution on the bottom layer of A-3D μPAD.

[0031] Figure 8 is the R / B value under different Co-CDs concentration (A) and OPD concentration (B) conditions in the Co-CDs / OPD system.

[0032] Fig. 9 Effects of different AAP concentrations (A), pH (B) and incubation time (C) on the ALP / AAP / Co-CDs / OPD system in A-3DμPAD.

[0033] Fig.10 The calibration curve of B / R ratio and ALP activity (Inset: different ALP activities (0-150UL -1 ) Corresponding image of A-3DμPAD paper chip).

[0034] Fig.11 The response of A-3DμPAD after adding interfering substances to the Co-CDs / OPD / AAP system (A) and the Co-CDs / OPD / ALP / AAP system (B).

[0035] Fig.12 The effect of different pH (A) and NaCl concentration (B) on the fluorescence intensity of Co-CDs. DETAILED DESCRIPTION

[0036] The technical solution in this embodiment will be described clearly and completely below in conjunction with the drawings in this embodiment. Obviously, the described embodiment is only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0038] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0039] Example 1

[0040] The preparation method of Co-doped carbon dot nanoparticles in this embodiment is as follows:

[0041] Weigh 10 mg of neutral red (NR, 3-amino-7-methylamino-2-methylphenazine hydrochloride), 10 mg of urea, and 20 mg of cobalt chloride (CoCl2), dissolve in 20 mL of ultrapure water, and transfer the mixture to a 50 mL stainless steel autoclave lined with Teflon (polytetrafluoroethylene), and heat at 120°C for 2 h (naturally cool at room temperature). The pink product is dialyzed (MWCO 500) for 2 days, filtered through a membrane (0.22 μm), and freeze-dried to obtain the Co-CDs product.

[0042] Comparative Example 1

[0043] Weigh 10 mg of neutral red (NR, 3-amino-7-methylamino-2-methylphenazine hydrochloride) and 10 mg of urea, dissolve in 20 mL of ultrapure water, and transfer the mixture to a 50 mL stainless steel autoclave lined with Teflon (polytetrafluoroethylene), and heat at 120°C for 2 h (natural cooling at room temperature). The product was dialyzed (MWCO 500) for 2 days, filtered through a membrane (0.22 μm), and freeze-dried to obtain a Co-free CDs product.

[0044] Example 2

[0045] The preparation method of Co-doped carbon dot nanoparticles in this embodiment is as follows:

[0046] Weigh 10 mg of neutral red (NR, 3-amino-7-methylamino-2-methylphenazine hydrochloride), 10 mg of urea, and 10 mg of cobalt chloride (CoCl2), dissolve in 20 mL of ultrapure water, and transfer the mixture to a 50 mL stainless steel autoclave lined with Teflon (polytetrafluoroethylene), and heat at 120°C for 2 h (naturally cool at room temperature). The pink product is dialyzed (MWCO 500) for 2 days, filtered through a membrane (0.22 μm), and freeze-dried to obtain the Co-CDs product.

[0047] Example 3

[0048] The preparation method of Co-doped carbon dot nanoparticles in this embodiment is as follows:

[0049] Weigh 10 mg of neutral red (NR, 3-amino-7-methylamino-2-methylphenazine hydrochloride), 10 mg of urea, and 5 mg of cobalt chloride (CoCl2), dissolve in 20 mL of ultrapure water, and transfer the mixture to a 50 mL stainless steel autoclave lined with Teflon (polytetrafluoroethylene), and heat at 120°C for 2 h (naturally cool at room temperature). The pink product is dialyzed (MWCO 500) for 2 days, filtered through a membrane (0.22 μm), and freeze-dried to obtain the Co-CDs product.

[0050] Please combine Figure 1 , (A) is the TEM image of Co-CDs prepared in Example 1 (inset: particle size distribution of Co-CDs); (B) is the XRD spectrum of Co-CDs prepared in Example 1; (C) is the UV-visible absorption spectrum and fluorescence excitation and emission spectrum of Co-CDs prepared in Example 1 (inset: images of Co-CDs under sunlight and 365nm UV lamp); (D) is the fluorescence spectrum of Co-CDs prepared in Example 1 at different excitation wavelengths (340-385nm).

[0051] exist Figure 1 In Figure A, the transmission electron microscope (TEM) image shows that the Co-CDs are spherical particles and are well dispersed with an average particle size of 3.25±0.64nm. The X-ray diffraction pattern of Co-CDs shows a diffraction peak at 2θ=26°, indicating the amorphous nature of the carbon atoms in Co-CDs ( Figure 1 B). In the UV-vis spectrum of Co-CDs ( Figure 1C), the absorption bands at 253nm and 276nm are the π-π* transitions of C=C, and the peaks at 343nm and 489nm are the n-π* transitions of C=O and C=N on the Co-CDs surface. Under 365nm excitation, Co-CDs emit strong red fluorescence at 609nm. The Co-CDs solution appears light pink under sunlight and shows bright red fluorescence under ultraviolet light (365nm). Figure 3 In D, when the excitation wavelength is between 340-385 nm, Co-CDs show non-excitation-dependent properties, indicating that Co-CDs have uniform nanostructures and luminescent centers. Using rhodamine B as a reference, the fluorescence quantum yield of Co-CDs was measured to be 13.8%.

[0052] Please combine Figure 2 , Figure 2 A is the FT-IR spectra of Co-CDs and CDs, Figure 2 B is the XPS spectra of Co-CDs and CDs, Figure 2 C is the high-resolution XPS spectrum of Co 2p of Co-CDs. Figure 2 D is the high-resolution XPS spectrum of O1s of Co-CDs. Figure 2 E is the high-resolution XPS spectrum of N1s of Co-CDs. Figure 2 F is the high-resolution XPS spectrum of C1s of Co-CDs.

[0053] Fourier transform infrared spectroscopy (FT-IR) of Co-CDs and CDs is shown in Figure 2. Figure 2 As shown in A, located at 3300, 2800, 1600, 1440 and 1052 cm -1 The characteristic peaks near the 527cm-1 peaks are attributed to the vibrations of OH / NH, CH, C=O / C=N, CN, and COC. The above results indicate that the surfaces of Co-CDs and CDs contain hydroxyl, amino, and carboxyl functional groups. Compared with CDs, Co-CDs have a -1 The absorption band at is attributed to the stretching vibration of Co-N, which indicates that the successful doping of cobalt (II) provides active sites for Co-CDs. X-ray photoelectron spectroscopy (XPS) was used to characterize the chemical composition and surface chemical state of Co-CDs. Figure 2As shown in Figure B, C, N and O element peaks were observed in both Co-CDs and CDs spectra, while an obvious Co 2p peak was observed in Co-CDs, proving the successful doping of Co element in Co-CDs. In the XPS spectrum of Co-CDs, the peaks at 780.90, 532.76, 399.58 and 285.55 eV are attributed to Co 2p (0.86%), O 1s (22.82%), N 1s (12.94%) and C1s (63.38%), respectively. The HR-XPS spectrum of Co 2p shows double peaks at 796.78 and 781.08 eV, and two corresponding satellite peaks appear at 786.28 and 802.78 eV, which are Co 2p 1 / 2 and Co 2p 3 / 2 , indicating that there is Co in the Co-CDs structure 2+ ( Figure 2 C) Figure 2 In D, the O 1s spectrum is decomposed into two peaks, corresponding to CO (533.58 eV) and C=O (532.28 eV). The high-resolution N1s spectrum shows three peaks at 400.88, 399.98 and 399.08 eV, indicating the presence of graphitic N, pyrrole N and pyridinic N ( Figure 2 E). The C 1s spectrum can be divided into three peaks located at 286.78, 285.88, and 284.88 eV, corresponding to C=O, CO / CN, and CC / C=C, respectively. Figure 2 F).

[0054] Oxidase-like activity of Co-CDs, OPD was selected as the chromogenic substrate. Figure 3 In A, the absorption signal in the Co-CDs / OPD system corresponds to the absorption peak of oxOPD, which is due to the Co-CDs catalyzing the reaction of OPD with O2 dissolved in H2O to generate oxOPD. Figure 3 As shown in B, the fluorescence spectrum of the Co-CDs / OPD system showed the characteristic emission of oxOPD at 565nm. Both UV-vis and fluorescence tests showed that Co-CDs had significant oxidase-like activity. Under the same conditions, CDs did not show oxidase-like activity to trigger the oxidation of OPD to produce oxOPD. These results verify that the oxidase-like activity of Co-CDs originates from the doped Co(Ⅱ). The catalytic activity of Co-CDs nanozymes also showed pH dependence similar to other oxidase-like enzymes. At pH 8.5, the oxidase-like activity reached its maximum ( Figure 3 C).

[0055] In addition, 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt [ABTS] and 3,3',5,5'-tetramethylbenzidine [TMB] were used to test the Co-CDs ( Figure 3 D and 3E). The Co-CDs+ABTS and Co-CDs+TMB systems had no characteristic absorption peaks at 417nm and 652nm, respectively. It may be that the electrostatic repulsion of negatively charged ABTS and the low solubility of TMB reduced the reaction ratio between the substrate and Co-CDs.

[0056] Electron spin resonance spectroscopy (ESR) was used to investigate the catalytic mechanism of Co-CDs, using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a free radical scavenger. Figure 3 As shown in F, the ESR spectrum of the Co-CDs / DMPO system has 6 obvious peaks, corresponding to DMPO-O2 · - adducts. This indicates that Co-CDs act as oxidase-like enzymes to activate dissolved O2 in H2O to produce a large amount of O2 ·- , and then oxidizes OPD to form oxOPD.

[0057] Please combine Figure 4 The A-3DμPADs-based alkaline phosphatase activity test strip in the embodiment of the present invention comprises a plasma separation membrane 1 (PSM) at the top, a developer layer 2 at the middle layer, and a color catalytic layer 3 at the bottom; the developer layer 2 comprises a hydrophobic substrate layer 21 and a first hydrophilic paper base region 22 loaded with a developer, and the first hydrophilic paper base region 22 is located below the plasma separation membrane 1; the color catalytic layer 3 comprises a hydrophobic substrate layer 31 and a second hydrophilic paper base region 32 loaded with Co-doped carbon dot nanoparticles (Co-CDs), Tris-HCl buffer and L-ascorbic acid-2-phosphate (AAP) prepared by any of the preparation methods described above; the first hydrophilic paper base region 22 and the second hydrophilic paper base region 32 are arranged to be connected.

[0058] The color developer is non-fluorescent o-phenylenediamine (OPD).

[0059] A hydrophobic annular region is also provided around the plasma separation membrane 1, and the hydrophobic annular region can be formed by spraying a hydrophobic oil film, or by opening holes on a hydrophobic substrate and pasting the plasma separation membrane 1 thereon.

[0060] The plasma separation membrane 1, the color developer layer 2 and the color catalyst layer 3 can be adhered together by double-sided tape.

[0061] Please combine Figure 4Specifically, in a specific example, the structure of the alkaline phosphatase activity detection test strip based on the A-3DμPADs mechanism is as follows:

[0062] The upper plasma separation membrane (PSM) is set with a sample area (6 mm in diameter) to separate plasma from whole blood;

[0063] The middle and bottom layers are filter paper (Whatman TM No.5), the reagent area diameter is 6 mm. In order to avoid direct reaction between Co-CDs and OPD, the color developer OPD (100 mM, 6 μL) was added dropwise to the middle layer reagent area (first hydrophilic paper base area 22), and dried at 25°C for 10 min. -1 , 6 μL) was added to the bottom reagent area (the second hydrophilic paper base area 32), dried at 25°C for 10 min, and L-ascorbic acid-2-phosphate (AAP) (75 mM, 6 μL) dissolved in Tris-HCl (10 mM, pH 8.5) was added in sequence and dried at 25°C for 10 min.

[0064] Please combine Figure 5 A-5B, ALP sensing mechanism is as follows: After adding ALP, ALP hydrolyzes L-ascorbic acid-2-phosphate AAP to generate ascorbic acid AA, and the oxidase-like properties of Co-CDs, which emit red fluorescence at 609nm, oxidize AA to generate DHAA. DHAA reacts with OPD to generate DFQ, which emits blue fluorescence at 430nm. In the absence of ALP, the generation of AA is prevented. Co-CDs catalyze the oxidation of OPD to oxOPD, which emits yellow fluorescence at 565nm. The characteristic absorption peaks of DFQ and oxOPD are also observed in the UV-vis spectrum ( Figure 5 C).

[0065] Please combine Figure 6 , based on the UV-visible spectrum, fluorescence lifetime and zeta potential test results. Figure 6 In A, the absorption peaks of DFQ and oxOPD are located at 330 and 420 nm, respectively, which overlap greatly with the excitation spectrum of Co-CDs at 365 nm. This indicates that the quenching of the fluorescence intensity of Co-CDs may be caused by IFE. Before and after the addition of DFQ and oxOPD, the fluorescence lifetime of Co-CDs remains unchanged ( Figure 6 B), indicating that there is no energy or electron transfer process between Co-CDs and DFQ or oxOPD. The zeta potentials of Co-CDs, DFQ, and oxOPD are -22.71, -18.65, and -24.4 mV, respectively. Figure 6C), electrostatic repulsion separates them from each other, hindering energy transfer. Therefore, it is feasible to apply Co-CDs nanozymes with fluorescence and oxidase-like properties to IFE-based ALP activity detection, which can produce obvious fluorescence color changes from red, yellow to blue.

[0066] In order to explain the best embodiment of A-3DμPAD-based ALP activity detection, reaction parameters such as reagent volume, concentration, incubation time and temperature were investigated. Figure 7 As shown in Figure 2, when the reagent volume is 18 μL, it can completely fill the sample area at the bottom of the A-3D μPAD without leaking out of the hydrophobic barrier. Figure 8 As shown, with the increase of Co-CDs concentration, the oxidase activity increased, resulting in an increase in the R / B ratio. When the OPD concentration was in the range of 25-125 mM, the R / B ratio also increased with the increase of OPD concentration.

[0067] The experimental results showed that the concentrations of Co-CDs and OPD were set at 1.0-1.5 mg mL -1 and 75-125mM; the detection conditions of alkaline phosphatase activity test paper are 50-100mM L-ascorbic acid-2-phosphate in a Tris-HCl environment with a pH of 8-9 and incubated at 37°C for 20-30min, both of which can achieve good experimental results.

[0068] Considering reagent consumption and sensitivity, the concentrations of Co-CDs and OPD were set to 1.25 mg mL -1 and 100mM. Fig. 9 The relationship between reaction conditions and B / R ratio was analyzed. The optimal condition for ALP detection was 75 mM AAP in Tris-HCl (10 mM, pH 8.5) and incubated at 37°C for 25 min.

[0069] A system for real-time detection of alkaline phosphatase activity in one embodiment of the present invention comprises an alkaline phosphatase test strip as described in any of the above items, a dark box and an electronic device, wherein the dark box is provided with an ultraviolet lamp, a placement area for placing the alkaline phosphatase test strip and a shooting hole arranged corresponding to the placement area, the electronic device is used to take a photo of the alkaline phosphatase test strip after color development through the shooting hole, and the electronic device is used to analyze and compare the photo of the alkaline phosphatase test strip after color development through software to obtain a detection result of whether alkaline phosphatase is detected.

[0070] Under the optimal experimental conditions described above, the performance of the portable A-3DμPAD for ALP fluorescence determination was verified. Fig.10 As shown, with the ALP activity (0-150U L -1) increases, the fluorescence color of the detection area changes from red and yellow to blue. By converting the color signal into the B / R ratio, the ALP activity and the B / R ratio are in the range of 0.5-150U L -1 The linear fitting equation is y=0.0073x+0.9350(R 2 0.9846), the detection limit (LOD) is 0.1UL -1 (S / N 3). The normal ALP level in the human body is maintained at 40-130U L -1 , clinical samples with high ALP activity should be diluted 3 times to make the ALP activity fall within the linear range of this method. Therefore, this method can fully meet the needs of clinical ALP monitoring. Compared with other reported ALP sensing strategies, the strategy established in this study showed high sensitivity, wider linear range and lower LOD (Table 1).

[0071] Table 1. Comparison of this method with reported ALP detection methods.

[0072]

[0073] Using common metal ions (Na + ,K + ,Ca 2+ Mg 2+ ), small molecules (L-glycine (L-Glycine, L-Gly); cholesterol (Chol); uric acid (UA); glucose (Glu)) and biomacromolecules (glucose oxidase (GOX); urate oxidase (UOX)) were used to evaluate the sensitivity and anti-interference ability of the Co-CDs / OPD system in detecting ALP. The concentrations of metal ions and small molecules were 100 μM, and the activity of biomacromolecules was 1000 U L -1 , ALP activity was set at 100 U L -1 .

[0074] like Fig.11 As shown in A, when ALP is added to the Co-CDs / OPD system, the R / B value increases significantly. When interfering substances are introduced into the Co-CDs / OPD / ALP / AAP system, the R / B ratio does not change much ( Fig.11 B). This indicates that the effects of these interfering substances on ALP detection are negligible. The excellent sensitivity and anti-interference ability are attributed to the specificity of ALP and the oxidase-like properties of Co-CDs. Under ultraviolet light irradiation (365nm) and high ionic strength, the fluorescence intensity of Co-CDs did not change significantly, indicating that Co-CDs have high stability ( Fig.12 ). These results indicate that the ALP / Co-CDs cascade system has great application potential in ALP detection in clinical disease diagnosis. In general, the excellent sensing performance, portable design, low cost and high-throughput analysis of A-3DμPAD provide an alternative platform for ALP detection.

[0075] In order to evaluate the feasibility of A-3DμPAD for ALP detection, whole blood samples were directly tested without pretreatment. As shown in Table 2, the test results of the above method were consistent with those of the biochemical detector in the laboratory, and the relative standard deviation (RSD) was less than 10%. The above results show that the method based on A-3DμPAD can be used for accurate and reliable quantitative analysis of ALP levels in actual samples.

[0076] sample <![CDATA[Biochemical Analyzer Detection in the Clinical Laboratory (UL -1 )]]> <![CDATA[The present invention detects (U L -1 )]]> RSD(%) 1 23.5 23.32±1.96 8.42 2 54.8 54.88±3.70 6.74 3 79.1 78.81±3.08 3.91 4 113.2 112.79±1.58 1.40 5 136.3 137.39±8.52 6.20 6 12.9 13.05±0.44 3.41 7 20.1 19.67±1.36 6.93 8 57.1 56.88±1.27 2.24 <![CDATA[9 a ]]> 240.9 246.75±6.96 2.82 <![CDATA[10 a ]]> 163.9 159.09±15.38 9.67

[0077] Similarly, the Co-CDs prepared in Example 2 and Example 3 were used to construct Co-CDs) / o-phenylenediamine (OPD, A-3DμPAD uric acid detection test strips, and also obtained good color development effects.

[0078] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing Co-doped carbon dot nanoparticles, characterized in that: Includes steps: S1, dissolving neutral red, urea and cobalt chloride in pure water at a weight ratio of 0.8-1.2:0.8-1.2:1.8-2.2 and a mass-liquid ratio of 1.8-2.2:1; S2, reacting the uniformly dispersed mixed solution in step S1 in a polytetrafluoroethylene-lined autoclave at 110° C. to 130° C. for 1.5 to 2.5 hours; S3, dialyzing the reaction solution in step S2 for 36-60 hours; S4, freeze-drying the solution obtained in step S3 to obtain Co-doped carbon dot nanoparticles.

2. The method according to claim 1, characterized in that The step S4 comprises filtering the solution obtained in step S3 through a filter membrane, and freeze-drying the filtered solution to obtain Co-doped carbon dot nanoparticles.

3. The method according to claim 2, characterized in that The membrane pore size of the filter membrane is 0.2-0.24 μm.

4. The method according to claim 1, characterized in that: The mass ratio of neutral red, chloroplatinic acid and ferric chloride in step S1 is 1:1:

2.

5. The method according to claim 1, characterized in that The reaction temperature in step S2 is 115° C. to 125° C., and the reaction time is 1.8-2.2 h; the dialysis time in step S3 is 45-55 h.

6. A Co-doped carbon dot nanoparticle, characterized in that: The method is prepared according to any one of claims 1 to 5.

7. Use of the Co-doped carbon dot nanoparticles as claimed in claim 6 for oxidizing ascorbic acid in real-time detection of alkaline phosphatase activity.

8. An alkaline phosphatase activity test strip, characterized in that: It includes a plasma separation membrane located at the top, a color developer layer located in the middle layer and a color catalytic layer located at the bottom; the color developer layer includes a first hydrophilic paper base region having a hydrophobic boundary and loaded with a color developer, and the first hydrophilic paper base region is located below the plasma separation membrane; the color catalytic layer includes a second hydrophilic paper base region having a hydrophobic boundary and loaded with Co-doped carbon dot nanoparticles and L-ascorbic acid-2-phosphate prepared by the preparation method described in any one of claims 1 to 5; the first hydrophilic paper base region and the second hydrophilic paper base region are arranged to be connected.

9. The alkaline phosphatase activity test strip according to claim 8, characterized in that: The color developer is non-fluorescent o-phenylenediamine; the concentrations of Co-doped carbon dot nanoparticles and o-phenylenediamine are set to 1.0-1.5 mg mL -1 and 75-125 mM; the detection conditions of the alkaline phosphatase activity test strip are 50-100 mM L-ascorbic acid-2-phosphate in a Tris-HCl environment with a pH of 8-9 and incubated at 37°C for 20-30 min.

10. A system for real-time detection of alkaline phosphatase activity, characterized in that: The invention comprises an alkaline phosphatase test strip, a dark box and an electronic device as described in any one of claims 8 to 9, wherein the dark box is provided with an ultraviolet lamp, a placement area for placing the alkaline phosphatase test strip and a shooting hole arranged corresponding to the placement area, the electronic device is used to take a photo of the alkaline phosphatase test strip after color development through the shooting hole, and the electronic device is used to analyze and compare the photo of the alkaline phosphatase test strip after color development through software to obtain a detection result of whether alkaline phosphatase is detected.

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